biochemical tests

#11: Hydrolysis Of Starch

• Medium: Starch Agar

• Substrate: Scratch 

• Enzyme: Amylase (exoenzyme) 

• Reagent: Iodine 

• End Product: Maltose 

  • Disaccharide composed of glucose 2

• Positive: Amber no starch

• Negative: Black starch is present

#12 Hydrolysis of Gelatin

• Medium: Gelatin

• Substrate: Gelatin 

• Enzyme: Gelatinase (Exoenzyme) 

• Reagent; n.a

• End product: Amino acids

• Positive: Liquid; produces gelatinase

  • Broke down into amino acids

• Negative: Solid; does not produce gelatinase. 

#13 Fermentation of Carbohydrates

• Medium: Phenol broth + protein 

  • Sugars will make acidic products

  • Protein will make basic products

• Substrates: Glucose, Sucrose, Lactose, Mannitol 

• Pathway: Glycolysis 

• Reagent: Phenol Red 

• End Product: Pyruvic acid, ATP, +/- CO₂ 

• Positive: Acid. Acid/Gas; only using sugars

• Negative: Magenta color like the control; only using proteins

#14 Reactions in Litmus Milk

• All mediums and reagents are Litmus milk 

• CO₂ is only formed in Fermentation reactions 

#14.A Fermentation Litmus Milk

• Substrate: Lactose

• Pathway: Fermentation

• If CO₂. glycolysis occurred 

• End products: Organic acids, ATP, +/- CO₂ 

• Positive: Pink milk, +/- hard curd. 1

#14.B Reduction of Litmus Milk

• Substrate: Litmus 

• Pathway: ETP

• End Products: leucolitmus (White/clear) at bottom plus ATp 

• Positive: white acidic milk, apple basic juice 

#14.C Alkalinization of Litmus Milk

• Substrate: Casein 

• Enzyme: Proteases 

• End products: Small peptides and ammonia 

• Positive: Blueberry milk 

#14.D Peptonization

• Substrate: Casein and lactalbumin 

• Enzyme: Proteases 

• End Products: Free amino acids and ammonia 

• Complete hydrolysis

  • Breaks down both proteins in milk 

• Positive: Grape/apple juice (Reduction occurs) +/- soft curd 

#14.E Curd Formation

• Substrate: Acidic or basic conditions

• Pathway: Fermentation or peptonization 

  • Fermentation: Hard curd 

  • Peptonization: Soft curd 

• End product: Curd 

• Positive: Hard or soft curd 

• Negative: No curd 

#15 Reduction of Nitrates

• Medium: Nitrate Broth 

• Substrate: KNO₃ 

• Pathway: ETP 

• Reagent: DAN, sulfuric acid 

• End product: ATP and KNO₂

• Positive: Magenta; can reduce to nitrite. 

• Negative: Any other color, like control or peach 

#16 Reduction of Catalase

• Medium: TSA plate/Microscope Slide 

• Substrate: Hydrogen Peroxide 

• Enzyme: Catalase 

• Reagent: Hydrogen Peroxide 

• End product: Oxygen bubbles and water 

• Positive: O₂ bubbles on colonies

• Negative: No bubbles 

#17 Production of Decarboxylase

• Medium: Decarboxylase Broth 

• Substrate: Arginine, Lysine, Ornithine 

• Enzyme: Decarboxylase 

• Reagent: Bromocresol Purple 

• End Product: Amine and Increase in CO₂ 

• Positive: Violet Color (basic) 

• Negative: Control or yellow (acidic) 

#18 Hydrolysis of Urea

• Medium: Urea Broth 

• Substrate: Urea 

• Enyzme: Urease 

• Reagent: Phenol Red 

• End Product: CO₂, NH₃ (ammonia) 

• Positive: Hot pink 

• Negative: Yellow to gold (acidic) 

#19 S.I.M.S Test

• S - Production of Hydrogen Sulfide

• Substrate: Cysteine, H₂S + FeSO₄

• Pathway: ETC

• End Products: H₂S + ATP; FeS + H₂SO₄ (iron sulfide + Sulfuric acid) 

• Positive: Black precipitate Iron Sulfide

#19 SIMS Test

• I - production of Indole

• Substrate: Tryptophan 

• Reagent: Kovacs

• End product: Indole, pyruvic acid, ammonia 

• Positive: Red ring

SIMS TEST

• Motility

Positive: Movement from the line

#20 MR.VP Test

Methyl-Red (MR)

• MR - Methyl Red 

• Medium: MR broth 

• Substrate: Glucose 

• Pathway: Glycolysis 

• Reagent: Methyl Red 

• End Product: ATP, Pyruvic Acid 

• Positive: Red; presence of strong acid (organic acids)

• Negative: Control, yellow basic; absence of strong acid 

#20 MR.VP Test

• Vogues Proskauer Test

• VP: Vogues Proskauer

• Medium: VP broth 

• Substrate: Glucose 

• Pathway: Alternative enzyme pathway 

• Reagent: D. Napthol, KOH Creatine

• End Product: ATP, AMC, Organic acids and alcohols

• Positive: Red ring on top 

#21 Utilization of Unusual Nitrogen (Ammonium Phosphate)

• Medium: Ammonium Phosphate Broth 

• Substrate: Ammonium Phosphate 

• Reagent: Bromcresol Purple 

• End Product: Ammonia and Phosphoric Acid 

• Positive: Yellow (acidic) 

• Negative: Burgundy/purple (basic) 

#22 Utilization of Unusual Carbon

• Medium:

  • Citrate Agar

• Substrate:

  • Citrate, ammonium 

• Enzyme: Citratase 

  • pH indicator: Bromthymol blue 

• End Product

  • Pyruvate, ammonia 

• Positive

  • Blue (basic)

• Negative

  • Green (acidic) 

#24 MSA-Mannitol Salt Agar

• Medium: MSA

• Substrate: Mannitol

• Pathway: Glycolysis 

• Reagent/pH indicator: Phenol Red 

• End Product: Organic acids, ATP 

• Positive: Yellow; agar turns yellow, acidic product.

• Negative: Red 

#25 Coagulase Production

• Medium: Rabbit Plasma and Sodium Citrate

• Substrate: Sodium Citrate 

• Enzyme: Coagulase

• End products: Clotted plasma 

• Positive: Liquid plasma = IT HAS COAGULASE. 

  • Increase in virulence  

• Negative: Solid media. 

#26 Hemolysin Production

• ALPHA

• Alpha: Incomplete

• Medium: Blood agar

• Substrate: RBC, partial hemoglobin, NO iron 

• Pathway: Incomplete 

• Positive: Greenish colonies 

  • The green colonies means that it doesn’t digest iron. 

  • Incomplete hemolysis. 

  • Examples 

    • Strep Pneumoniae

    • Strep Aureus. 

#26 Hemolysin Production

• BETA

• Beta: Complete 

• Medium: Blood agar

• Substrate: RBC, Hemoglobin, plasma protein, iron 

• Pathway: Complete 

• Positive: Clear zones 

  • Examples:

    • Strep equi

    • S. Bovis. 

#26 Hemolysin Production

• GAMMA

• Medium: Blood agar

• Substrate: plasma proteins

• Positive: Red agar 

• Pathway: incomplete 

  • It only goes after the plasma proteins

• Staph. Epidermidis 

#27 Latex Agglutination

• Medium: Latex beads coated with antibodies against coagulase 

• Positive: clumps

• Negative: no clumps 

#33 Zone of Inhibition

• Clear zone around the discs

  • Increase diameter: effective at inhibiting growth

  • Decrease diameter: not effective at inhibiting growth

Which is positive and negative for Hydrolysis of Starch and why?

B. Sub is positive while E. Coli is negative.

• B Sub produces amylase which produces an amber color making it positive.

• E. Coli is DOES NOT produce amylase which means that it is negative( black color)

What does a POSITIVE test of Hydrolysis of Gelatin look like?

Liquid media.

• This means that it produces GELATINASE, which breaks down amino acids.

What does a NEGATIVE Hydrolysis of Gelatin Test look like?

Solid media.

• This means that it DOES NOT PRODUCE Gelatinase.

Is this POSITIVE or NEGATIVE for #16 Reduction of Catalase?

This is Positive.

• Since it produced bubbles, it is positive for Catalase.

What does it mean if a Catalase Test is negative?

There would be no bubbles and Catalase negative.

What type of bacteria can produce Catalase?

Aerobic and Facultative Anaerobic.

What type of bacteria cannot produce Catalase?

Obligate Anaerobes.

What does a positive and negative test mean for Hydrolysis of Urea?

• Positive: HOT PINK

  • BASIC

    • Produces UREASE

• Negative: YELLOW - GOLD `

  • ACIDIC

    • Does NOT produce UREASE

What does a POSITIVE and NEGATIVE test mean for Fermentation of Carbohydrates?

• Positive

  • ACID. ACID/GAS

    • Produces a yellow color because it only uses sugars.

• Negative

  • BASIC

    • MAGENTA color like control.

      • Only uses proteins.

What does a POSITVE and NEGATIVE test mean for Reduction of Nitrates?

• Positive

  • Magenta color

    • This means that Nitrate can reduce to Nitrite.

• Negative

  • Control/Peach color

    • This means that it does not reduce Nitrate to Nitrite.

What does a Positive and Negative Test mean for Production of Decarboxylase?

• Positive

  • Violet Color

    • Produces Decarboxylase making it BASIC.

• Negative

  • Control or Yellow

    • Does not produces Decarboxylase, making it ACIDIC.

What does a positive and negative test mean for Ammonium Phosphate?

• Positive

  • Yellow (acidic)

    • NH₃ is metabolized, remaining phosphate is yellow

• Negative

  • Purple (basic)

    • NH₃ is not metabolized, remaining basic.

What are the following results for the Litmus Milk Tests?

• Fermentation

  • Positive: PINK MILK, +/- hard curd

• Reduction

  • Positive: White acidic milk, apple basic juice

• Alkanization

  • Positive: Blueberry milk

• Peptonization

  • Positive: Grape/apple juice, +- soft curd

• Curd Formation

  • Positive: Hard or soft curd

  • Negative: No curd

What are the following results for SIM tests?

• S - Hydrogen Sulfide

  • Positive: Black Precipitate

    • Hydrogen Sulfide reacts with Iron Sulfate resulting in Iron Sulfide,.

• I - Production of Indole

  • Positive: Red Ring

    • Kovac Reagent is used to determine positivity for indole.

• M - Motility

  • Positive: Movement from the line.

What does a positive and negative test mean for the MRVP tests?

• MR

  • Positive: RED

    • Presence of strong acid meaning it can ferment carbohydrates

  • Negative: Control/Yellow

    • There is no acids.

• VP

  • Positive: RED RING ON TOP

    • This indicates the presence of AMC.

What does a positive and negative test mean for Sodium Citrate test?

• Positive:

  • Blue (BASIC)

    • It can metabolize citrate.

• Negative:

  • Green (ACIDIC)

    • No metabolization of citrate

What does a positive test of the MSA Salt Agar test indicate?

Positive Test indicates that the bacteria is pathogenic.

• If it is acidic, this means that is it pathogenic because acidic products were formed.

What does a negative test of the MSA Salt agar plate indicate?

It indicates that it is not pathogenic and doesn’t produce acidic products.

What does a positive and negative test for Coagulase production indicate?

• Positive

  • Solid Media

    • this means that it has COAGULASE, and increases it virulence.

• Negative

  • Liquid Media

    • Does not have COAGULASE .

This is an Alpha Hemolysin Test, what does this test indicate?

• Contains green colonies which means that it does NOT digest IRON. Its pathway is incomplete.

This is a BETA HEMOLYSIN test, what does this indiciate?

• Contains see-through zones which means that it digests IRON. Its pathway is complete.

This is a Gamma HEMOLYSIN test, what does this indicate?

• The red agar indicates that it only goes after the plasma proteins. Its pathway is incomplete.

This is a Latex Agglutination Test, what does this indicate?

• Positive

  • Clumps

    • There is coagulase

• negative

  • No clumps

    • There is no coagulase.

This is a Zone of Inhibition Test, what does this indicate?

• Increase in Diameter - Effective at inhibiting growth

• Decrease in Diameter - Not effective at inhibiting growth

If the hydrolysis of starch is positive, it indicates the presence of what enzyme?

Amylase.

If a microorganism can hydrolyze gelatin, it will convert it to

amino acids.

If a microorganism can ferment carbohydrates, what end products are produced?

Pyruvic acid, ATP, and CO₂

The pH indicator used in the fermentation of carbohydrates is____, and turns _____ under acidic conditions.

Phenol Red and turns to Yellow.

Name the 2 Reagents used in the Reduction of Nitrates Test.

DAN and Sulfuric Acid.

Catalase converts hydrogen peroxide into

Oxygen bubbles and water.

The indicator used in the production of carboxylases is?

Brom Cresol Purple.

The Decarboxylase test is done under aerobic or anaerobic conditions?

Anaerobic conditions.

A positive test for urease would change the pH indicator to what color?

Hot pink.

What 3 tests can be done with the SIMS reactions?

• production of hydrogen sulfide

• production of indole

  • motility test

The substrate used in the MR-VP Test can be converted

• Substrate: Glucose

  • Converted to ATP and Pyruvic acid in MR

  • Converted to ATP, AMC, Organic acids and alcohols in VP.

The reagent used for the VP test is

• Alpha Napthol

• Potassium Hydroxide Creatine.

MSA is plate is considered both a selective and differential media plate, why is that?

• Selective

  • Staph grows because this is a high salt concentration

• Differential

  • Distinguishes between pathogenic and nonpathogenic.

Can a staphylococcus organism that is found to be pathogenic, also be considered virulent?

No, pathogenic doesn’t mean virulent.

• Pathogenic means that it can cause disease.

• Virulent is the degree of how dangerous a pathogen can be.

What two tests can be done for the presence of coagulase?

• #26 Rabbit Plasma Test

  • Coagulase Production

• #27 Latex Agglutination

What tests can be used to determine virulence?

• Coagulase

• Latex Agluttination

• Hemolysin

In the Litmus milk test, an Apple juice/Consistency means that

• Peptonization

  • acidic products

• Reduction

  • losing color

Fermentation means

acidic products

peptonization means

complete hydrolysis.

• Breaks down both proteins in milk

Alkalinization means

Incomplete hydrolysis, only breaking down one protein.

Gas production only happens

during FERMENTATION reactions.

White hard curd with fissures indicates

• White caused by

  • Reduction

• Fissures caused by

  • Gas Production

• Hard Curd caused by

  • Ferment

Rennet Curd indicates

• Soft Curd

  • only happens during Peptonization

Leucolitmus indicates

• Bleached milk

  • Reduction

Blueberry Shake indicates

• Alkanization only.

  • incomplete hydrolysis

Pink milk indicates

Fermentation only.

White hard curd indicates

Reduction and fermentation.

Grape juice indicates

Peptonization,

Pink hard curd

Fermentation only.

Nitrate is reduced to ______, during the Reduction of Nitrates Test.

Nitrite. KNO₃ → KNO₂

What are the tests that are pH dependent?

• Decarboxylase

• Urea Test

Red blood cells are broken down in

• Alpha and Beta Hemolysis

Partial breakdown of the hemoglobin occurs in

Alpha Hemolysis

Iron is broken down in

Beta hemolysis

Complete breakdown of hemoglobin occurs in

Beta hemolysis

Breakdown of plasma proteins occurs in

Gamma and Beta hemolysis.

Obligate Aerobe grows in

Tall and Slant

Obligate anaerobes grow in

Thioglycollate

Facultative Anaerobe grows in

Tall, slant, thioglycollate.

Microaerophile grows in

Thioglycollate only on top and in the tall on the stab line.

The ammonium phosphate test products what 2 positive end products?

ammonia and phosphoric acid.

What is the pH indicator used in ammonium phosphate?

Brom cresol purple.

What color will Brom Cresol Purple change to in acidic conditions due to the ammonia phosphate test?

Yellow acidic products

A positive test for sodium citrate would be

Turbidity

What is the Medium for Hydrolysis of starch?

Agar plate

• Substrate: Starch

What is the medium for Hydrolysis of Gelatin?

Gelatin

• Substrate: Gelatin

What is the medium for fermentation of carbs?

Phenol + protein both

• Substrate: Glucose, mannitol, sucrose, sucrose.

What is the medium for all litmus milk tests?

Litmus milk

What is the medium for Reduction of Nitrates?

Nitrate broth

• Substrate: KNO₃ Potassouim Nitrate